1. Field of the Invention
This invention relates to constructs comprising three antimicrobial domains each harboring a unique lytic activity: a CHAP endopeptidase, an amidase, and a glycyl-glycine endopeptidase. In one embodiment, the CHAP endopeptidase and the amidase are provided by the peptidoglycan hydrolase, LysK endolysin, and the glycyl-glycine endopeptidase is provided by another peptidoglycan hydrolase, Lysostaphin generating a pathogen-specific triple fusion construct. The LysK endolysin specifically attacks the peptidoglycan cell wall of untreated, live staphylococci including S. aureus and methicillin-resistant Staphylococcus aureus (MRSA); Lysostaphin is a potent anti-staphylococcal bacteriocin. The constructs comprising the three functional antimicrobial domains are designed to be refractory to resistance development and can be used to treat staphylococcal pathogens including multi-drug resistant strains MRSA and USA300.
2. Description of the Relevant Art
S. aureus is an opportunistic bacterial pathogen responsible for a diverse spectrum of human and animal diseases. Although S. aureus may colonize mucosal surfaces of healthy humans, it is also a major cause of wound infections and has the invasive potential to induce severe infections, including osteomyelitis, endocarditis, and bacteremia with metastatic complications (Lowy, F. D. 1998. New England J. Med. 339: 520-532). Coagulase-negative staphylococci (CoNS) and S. aureus are the most common pathogens in nosocomial bacteremias and infections of implanted devices (Gordon et al. 2001. Ann. Thorac. Surg. 72: 725-730; Malani et al. 2002. Clin. Infect. Dis. 34: 1295-1300. Although methicillin-resistant S. aureus (MRSA) has classically been regarded as a nosocomial pathogen, it has emerged as a cause of community-acquired infections in hosts without predisposing risk factors. Superficial skin and soft tissue infections caused by MRSA are increasingly seen in clinical practice. There are limited treatment options available in terms of topical antimicrobial agents, and some strains of MRSA have developed resistance to topically applied antimicrobial agents. MRSA account for 40%-60% of nosocomial S. aureus infections in the U.S., and many of these strains are multi-drug resistant. Recent data indicate that more patients in U.S. hospitals die from MRSA (>18,000 per year) than AIDS (Klevens et al. 2007. JAMA 298: 1763-1771). MRSA strains with reduced susceptibility or resistance to vancomycin have also been reported (Zhu et al. 2008. Antimicrob. Agents Chemother. 52: 452-457). Because S. aureus cannot always be controlled by antibiotics and because MRSA isolates are becoming increasingly prevalent in the community, additional control strategies are sorely needed.
Peptidoglycan is the major structural component of the bacterial cell wall and can be up to 40 layers thick. Bacteria have autolytic peptidoglycan hydrolases that allow the cell to grow and divide. Another well-studied group of peptidoglycan hydrolase enzymes are the bacteriophage (viruses that infect bacteria) endolysins. Endolysins allow the phage to escape from the bacterial cell during the phage lytic cycle. Some Gram-positive bacteria exposed to purified phage lysins externally undergo exolysis or “lysis from without.” Use of phage endolysins as antimicrobials has not been reported for treatment of Gram-negative bacteria, presumably due to the presence of an outer membrane that prevents access to the peptidoglycan (Loessner, M. J. 2005. Curr. Opin. Microbiol. 8: 480-487). Peptidoglycan is unique to bacteria and has a complex structure (Loessner, supra) with a sugar backbone of alternating units of N-acetyl glucosamine (GN) and N-acetylmuramic acid (MN). Each MN residue is amide linked to a short pentapeptide chain. Characteristic of S. aureus is the pentaglycine bridge that connects the L-Lys of the stem peptide to the D-Ala at position 4 of a neighboring subunit (FIG. 1). Peptidoglycan hydrolases have evolved a modular design to deal with this complexity. Although single domain endolysins can lyse the target pathogen (Sanz et al. 1996. Eur. J. Biochem. 235: 601-605), endolysins can also harbor two short domains (˜100-200 amino acids), each encoding a different peptidoglycan hydrolase activity.
Three classes of peptidoglycan hydrolase domains have been identified: endopeptidases, amidases, and glycosidases (includes glucosaminidase and lysozyme-like muramidases) (Lopez and Garcia. 2004. FEMS Microbiol. Rev. 28: 553-580; FIG. 1). Alignment of conserved domain sequences from multiple peptidoglycan hydrolase proteins has identified non-variant amino acid positions that, when mutated, can destroy the hydrolytic activity of the domain (Pritchard et al. 2004. Microbiology 150: 2079-2087; Huard et al. 2003. Microbiology 149: 695-705; Bateman and Rawlings. 2003. Trends Biochem. Sci. 28: 234-237; Rigden et al. 2003. Trends Biochem. Sci. 28: 230-234). Chimeric peptidoglycan hydrolases have been created by the exchange of cell wall binding domains of two lysins (Croux et al. 1993. Mol. Microbiol. 9: 1019-1025). Enzymatic activity was retained and regulatory properties exchanged when the cell wall binding domains of choline-binding pneumococcal and clostridial lysins were swapped. Intra-generic chimeric fusion lysins are also functional (Diaz et al. 1990. Proc. Natl. Acad. Sci. USA 87: 8125-8129).
Lysostaphin is a bacteriocin secreted by S. simulans, that lyses S. aureus (Browder et al. 1965. Biochem. Biophys. Res. Commun. 19: 389). The endopeptidase activity is specific to the glycyl-glycyl bonds of the staphylococcal peptidoglycan inter-peptide bridge (FIG. 1). It is known that Lysostaphin can kill planktonic S. aureus (Walencka et al. 2005. Pol. J. Microbiol. 54: 191-200; Wu et al. 2003. Antimicrob. Agents Chemother. 47: 3407-3414), as well as MRSA (Dajcs et al. 2000. Am. J. Opthalmol. 130: 544), vancomycin-intermediate S. aureus (Patron et al. 1999. Antimicrob. Agents Chemother. 43:1754-1755), and other antibiotic-resistant strains of S. aureus (Peterson et al. 1978. J. Clin. Invest. 61: 597-609). Lysostaphin can also kill S. aureus growing in a biofilm (Walencka, supra; Wu, supra), and it exhibits limited activity against CoNS (Cisani et al. 1982. Antimicrob. Agents Chemother. 21: 531-535); McCormick et al. 2006. Curr. Eye Res. 31: 225-230).
S. simulans produces Lysostaphin and avoids its lytic action by the product of the Lysostaphin immunity factor (lif) gene [same as endopeptidase resistance gene (epr) (DeHart et al. 1995. Appl. Environ. Microbiol. 61: 1475-1479) that resides on a native plasmid (pACK1) (Thumm and Gotz. 1997. Mol. Microbiol. 23: 1251-1265). The lif gene product functions by inserting serine residues into the peptidoglycan cross bridge, thus interfering with the ability of the glycyl-glycyl endopeptidase to recognize and cleave this structure. Similarly, mutations in the S. aureus femA gene (factor essential for methicillin resistance) (Sugai et al., 1997. J. Bacteriol. 179: 4311-4318) result in a reduction in the peptidoglycan interpeptide cross bridge from pentaglycine to a single glycine, rendering S. aureus resistant to the lytic action of Lysostaphin. MRSA have been shown to mutate femA when exposed in vitro or in vivo to sub-inhibitory doses of Lysostaphin (Climo et al. 2001. Antimicrob. Agents Chemother. 45: 1431-1437).
Grundling et al. identified lyrA (Lysostaphin resistance A) that, when mutated by a transposon insertion, reduced S. aureus susceptibility to Lysostaphin (Grundling et al. 2006. J. Bacteriol. 188: 6286-6297). Although some structural changes were noted in peptidoglycan purified from the mutant, the purified peptidoglycan was susceptible to Lysostaphin and the phi11 endolysin, suggesting that changes in accessibility of the enzyme to its substrate may have rendered the strain Lysostaphin resistant.
Bacterial resistance to antibiotics usually involves the acquisition of enzymes that 1) inactivate the antibiotic; 2) reduce membrane permeability; 3) facilitate active efflux of the antimicrobial from the cell; 4) modify the target protein to a resistant form; or 5) produce higher quantities of the target protein. Alternatively, the original target protein can be 6) altered via a mutational or recombination event at the endogenous gene to an antibiotic-resistant form; or 7) the organism can be protected through the multi-faceted changes that accompany growth in a biofilm (Spratt, B. G. 1994. Science 264: 388-393).
The Gram-positive peptidoglycan is on the cell surface, outside of the cell membrane. Many mechanisms of resistance development take advantage of the ability to inactivate the antimicrobial inside the cell. Targets outside the cytoplasmic membrane reduce the possible mechanisms by which resistance can emerge (Spratt, supra). Although there have been no reports of extracellular inactivation of peptidoglycan hydrolase enzymes, S. aureus does secrete proteases that might degrade peptidoglycan hydrolases. A regulatory mutation that increases the activity, synthesis, regulation, or secretion of staphylococcal proteases (such as sarA (Karlsson et al. 2001. Infect. Immun. 69: 4742-4748) might confer some level of resistance. Similarly, although phi11 and Lysostaphin could digest purified lyrA peptidoglycan, this mutant is slightly resistant to Lysostaphin, suggesting that resistance mechanisms could exist due to changes in surface structures that limit accessibility to the target peptidoglycan (Grundling, supra). O-acetylation of peptidoglycan N-acetyl muramic acid residues by an O-acetyltransferase (OatA) results in resistance to human lysozyme and correlates with heightened virulence of some S. aureus strains (Bera et al. 2006. Infect. Immun. 74: 4598-4604).
Bacteriophage endolysins are relatively new antimicrobials compared to Lysostaphin, which was described in the 1960's (Browder, supra). Despite repeated attempts, no strains of bacteria that can resist lysis by bacteriophage endolysins have been reported (Loeffler et al. 2001. Science 294: 2170-2172: Schuch et al. 2002. Nature 418: 884-889; Fischetti, V. A. 2005. Trends. Microbiol. 13: 491-496). Bacteriophages and bacteria may have evolved such that phages have selected immutable target peptidoglycan bonds for cleavage with the endolysin to guarantee escape from the bacterium.
The near-species specificity of phage lysins avoids many pitfalls associated with broad range antimicrobial treatments. Broad range antimicrobials lead to selection for resistant strains, not just in the target pathogen, but also in co-resident commensal bacteria exposed to the drug. The acquisition of antibiotic resistance is often accomplished by transfer of DNA sequences from a resistant strain to a susceptible strain. This transfer is not necessarily species or genus limited, and can lead to commensal bacteria that are both antibiotic resistant and that can serve as carriers of these DNA elements for propagation to neighboring bacteria. Those neighboring strains (potential pathogens) with newly acquired resistance elements can emerge as antibiotic resistant strains during future treatment episodes. Thus, in order to reduce the spread of antibiotic resistance, it is recommended to avoid subjecting commensal bacterial communities to broad range antibiotics. Toward this end, FDA, USDA, and CDC promote the development of antimicrobials that reduce the risk of resistance development (CDC Action Plan: Retrieved from the Internet: .cdc.gov/druq resistance/actionplan).
Endolysins with two active domains are expected to be more refractory to resistance development since the cell will need to mutate or modify multiple target bonds to resist the lytic action of two activities (Fischetti, supra). The use of two bacteriophage endolysins has been reported to have a synergistic effect in the killing of streptococcal pathogens both in vitro (Loeffler et al. 2003. Antimicrob. Agents Chemother. 47: 375-377) and in vivo in a mouse sepsis model (Jado et al. 2003. J. Antimicrob. Chemother. 52: 967-973). This is consistent with synergy and better cure rates observed in models of S. aureus infections in which animals are treated with either antibiotics or Lysostaphin plus an antibiotic (Climo et al. 1998. Antimicrob. Agents Chemother. 42: 1355-1360; Climo et al. 2001, supra). Synergistic bactericidal activity has also been demonstrated with an endolysin and an antibiotic against S. pneumoniae (Djurkovic et al. 2005. Antimicrob. Agents Chemother. 49: 1225-1228).). A recent patent application (Kokai-Kun, J. F. 2003. US 20030211995) indicates there is synergy with Lysostaphin and the phi11 endolysin or the antibiotic bacitracin against S. aureus. 
Lysostaphin or endolysin injections can cure bacterial infections and do not raise an adverse immune response. It has been reported that Lysostaphin was efficacious in treating S. aureus animal infections, but the preparation was likely contaminated with other bacterial antigens, and actual doses were probably less than those described in the 1960s (reviewed in (Climo et al. 1998, supra). Lysostaphin has also been used to treat bovine mastitis (Oldham and Daly. 1991. J. Dairy Sci. 74: 4175-4182). The treatment effectively cleared the milk of S. aureus, and no deleterious effects to the animals were reported. Nonetheless, the majority of Lysostaphin-treated quarters relapsed after treatment ceased.
Peptidoglycan hydrolases have been proposed for human antimicrobial applications (Fischetti, V. A. 2003. Ann. N.Y. Acad. Sci. 987: 207-214; Fischetti 2005, supra; Schuch et al., supra), and they have demonstrated efficacy in animal models for eliminating Group B streptococcal colonization (Cheng et al. 2005. Antimicrob. Agents Chemother. 49: 111-117; Nelson et al. 2001. Proc. Natl. Acad. Sci. USA 98: 4107-4112), pneumococcal sepsis (Jado et al., supra), and S. aureus infection of mammary glands in transgenic mice (Kerr et al. 2001. Nat. Biotechnol. 19: 66-70) and cows (Wall et al. 2005. Nat. Biotechnol. 23: 445-451). Lysostaphin significantly increased survival of neonatal rat pups when given intravenously (IV) at either 30 or 60 min post S. aureus challenge (Oluola et al. 2007. Antimicrob. Agents Chemother. 51: 2198-2200). In a recent catheter-induced S. aureus endocarditis model, Lysostaphin was tolerated by the systemic route with minimal adverse effects (Climo et al. 1998, supra). Rabbits injected weekly with Lysostaphin (15 mg/kg) for 9 wks by the IV route produced serum antibodies that resulted in an eight-fold reduction in its lytic activity, consistent with earlier work (Schaffner et al. 1967. Yale J. Biol. Med. 39: 230-244), but no adverse immune response. It is believed that high purity and the absence of Gram-negative lipopolysaccharide are essential for guaranteeing a minimal host immune response.
Serum antibodies raised to phage endolysins specific to Bacillus anthracis, Streptococcus pyogenes, or Streptococcus pneumoniae slowed but did not block in vitro killing of the organism in vivo (Fischetti 2005, supra; Loeffler et al. 2003, supra). Cpl-1, a S. pneumoniae-specific phage lysin, was injected IV 3 times per week into mice for 4 wks, and 5 of 6 mice tested positive for IgG antibodies to Cpl-1. Vaccinated and naive mice were then challenged IV with pneumococci, and the mice were treated IV with 200 μg Cpl-1 after 10 h. Bacteremia was reduced within 1 min to the same level in both mouse groups, indicating that the antibody did not neutralize the enzyme in vivo (Loeffler et al. 2003, supra). Western blot analysis revealed that Cpl-1 and Pal elicited antibodies 10 d after a 200-μg injection in mice, but the second injection (at 20 d) also reduced the bacteremia profile 2-3 log units, indicating that the antibodies were not neutralizing in vivo. All mice recovered fully with no apparent adverse side effects or anaphylaxis (Jado et al. 2003, supra). A bacteriophage lysin also cleared streptococci from the blood of rats in an experimental endocarditis model, although antibody production was not monitored in this study (Entenza et al. 2005. Infect. Immun. 73: 990-998). Similarly, aqueous preparations of phage lysins have been proposed for the control of pathogenic bacteria on human mucous membranes (Fischetti 2003, supra) and mucosal clearing has been obtained with phage lytic enzymes applied to the murine vagina, oropharynx (Cheng et al. 2005, supra), and oral cavity (Nelson et al., supra). The mucosal immune response to these enzymes was not monitored in any of these studies.
Thus, S. aureus is a significant pathogen in both agricultural and human disease. Multi-drug resistant strains have become more prevalent, especially nosocomial and community acquired strains, and current antibiotic treatments are often less than 50% effective. This increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials that are refractory to resistance development.